Rameshwar Sharma

The Tomato Sequencing Project, the First Cornerstone of the International Solanaceae Project (SOL)

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the ‘International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation’. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae—and beyond—achieves a context that will facilitate a systems biology approach.

Anthocyanin-DNA copigmentation complex: mutual protection against oxidative damage

The absorption spectra of a cyanidin derivative showed a 15–20 nm bathochromic shift when mixed with calf thymus DNA (ctDNA), indicating formation of a cyanidin-DNA copigmentation complex. Exposure of either cyanidin or ctDNA individually to hydroxyl radicals (OH) obtained in the Fenton reaction between ferrous ions and hydrogen peroxide caused severe oxidative damage. However, formation of cyanidin-DNA complex prior to exposure to OH• protected both the cyanidin and ctDNA from the oxidative damage. These results suggest that cyanidin-DNA copigmentation might be a possible defense mechanism against oxidative damage of DNA and may have in vivo physiological function attributable to the antioxidant ability of anthocyanins.

Antioxidant ability of anthocyanins against ascorbic acid oxidation

Oxidation of ascorbic acid by metal ions like copper was prevented by addition of anthocyanin (cyanidin derivative). Previous reports attributed the ability of flavonoids to protect ascorbate oxidation to the metal complexing properties of these compounds. We provide evidence that anthocyanin not only chelates metal ions, but also forms an ascorbic acid (copigment)-metal-anthocyanin complex, which could be the possible protection mechanism.

Ultraviolet-B-Responsive Anthocyanin Production in a Rice Cultivar Is Associated with a Specific Phase of Phenylalanine Ammonia Lyase Biosynthesis

Seedlings of 17 rice (Oryza sativa L.) cultivars were classified on the basis of anthocyanin pigmentation into three groups: an acyanic group with 9 cultivars, a moderately cyanic group with 5 cultivars, and a cyanic group with 3 cultivars. Seedlings of the cyanic group were deep purple in color, possessing copious amounts of anthocyanin in shoots. Sunlight (SL)-mediated anthocyanin and phenylalanine ammonia lyase (PAL) induction in a cyanic cultivar, purple puttu, was compared with an acyanic cultivar, black puttu. A brief exposure of dark-grown purple puttu seedlings to SL induced anthocyanin formation during a subsequent dark period with a peak at 24 h. The magnitude of SL-mediated anthocyanin induction is age dependent, the 4-d-old seedlings being the most responsive to SL. The anthocyanin induction in purple puttu seedlings is mediated exclusively by the ultraviolet-B (UV-B) component of SL. The SL-triggered anthocyanin induction was reduced by about 30% by a terminal far-red light pulse and was restored by a red light pulse, indicating the role of phytochrome in modulation of anthocyanin level. The SL-mediated induction of PAL showed two peaks, one at 4 h and the other at 12 h. Whereas the first PAL peak (4 h) was induced by phytochrome and was seen in both cultivars, the second PAL peak (12 h) was inducible by UV-B only in the cyanic purple puttu cultivar.

Purification and characterization of uv-b induced phenylalanine ammonia-lyase from rice seedlings

On exposure of rice seedlings to UV-B radiations, a four fold induction of PAL activity was observed. Purification of induced PAL revealed the presence of a single isoform with a molecular mass 320 kD and a subunit Mr of 84 kD. The enzyme exhibited Michaelis-Menten kinetics with a Km of 500 μM, optimal pH 8.9 and an activation energy 1.9 kcal. The induced enzyme was sensitive to inhibition by phenylpropanoid intermediates involved in flavonol and anthocyanin biosynthetic pathway.

Chromoplast-Specific Carotenoid-Associated Protein Appears to Be Important for Enhanced Accumulation of Carotenoids in hp1 Tomato Fruits

A chromoplast protein may assist in the sequestration and stabilization of carotenoids. Tomato (Solanum lycopersicum) high-pigment mutants with lesions in diverse loci such as DNA Damage-Binding Protein1 (high pigment1 [hp1]), Deetiolated1 (hp2), Zeaxanthin Epoxidase (hp3), and Intense pigment (Ip; gene product unknown) exhibit increased accumulation of fruit carotenoids coupled with an increase in chloroplast number and size. However, little is known about the underlying mechanisms exaggerating the carotenoid accumulation and the chloroplast number in these mutants. A comparison of proteome profiles from the outer pericarp of hp1 mutant and wild-type (cv Ailsa Craig) fruits at different developmental stages revealed at least 72 differentially expressed proteins during ripening. Hierarchical clustering grouped these proteins into three clusters. We found an increased abundance of chromoplast-specific carotenoid-associated protein (CHRC) in hp1 fruits at red-ripe stage that is also reflected in its transcript level. Western blotting using CHRC polyclonal antibody from bell pepper (Capsicum annuum) revealed a 2-fold increase in the abundance of CHRC protein in the red-ripe stage of hp1 fruits compared with the wild type. CHRC levels in hp2 were found to be similar to that of hp1, whereas hp3 and Ip showed intermediate levels to those in hp1, hp2, and wild-type fruits. Both CHRC and carotenoids were present in the isolated plastoglobules. Overall, our results suggest that loss of function of DDB1, DET1, Zeaxanthin Epoxidase, and Ip up-regulates CHRC levels. Increase in CHRC levels may contribute to the enhanced carotenoid content in these high-pigment fruits by assisting in the sequestration and stabilization of carotenoids.

The polycotyledon Mutant of Tomato Shows Enhanced Polar Auxin Transport

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively regulates PAT in tomato and its contribution to plant development and organogenesis.

Amylases synthesis in scutellum and aleurone layer of maize seeds

The endosperm of germinating maize seeds contains four isozymes of alpha-amylases (alpha-amylase-1 to -4) and one isozyme of beta-amylase. The alpha-amylases were purified by affinity chromatography on amylose and separated by DEAE-cellulose chromatography, into two groups, namely alpha-amylases-1,2 and alpha-amylases-3,4; and beta-amylase was purified by precipitation as a glycogen-enzyme complex. The molecular weight of alpha-amylases-1 and -2 was 46 kD, alpha-amylases-3 was 44.5 kD and of alpha-amylases-4 was 47.5 kD. The molecular weight of beta-amylase was 56 kD. During seed germination increase in amylolytic activity in endosperm was mainly contributed by secretion of alpha-amylases from adjoining aleurone layer and scutellum. The synthesis and secretion of alpha-amylases was first initiated in the scutellum followed by aleurone layers. Exogeneous Ca2+ stimulated synthesis of alpha-amylases in both eleurone layer and scutellum. In contrast, though scutellum and aleurone layer synthesized beta-amylase but it was not secreted to the medium. These results suggest that during the early germination period, alpha-amylases secreted from scutellum mobilizes starch.

Differential expression and properties of phenylalanine ammonia-lyase isoforms in tomato leaves

In tomato leaves, excision and light treatments increase phenylalanine ammonia-lyase activity, which is contributed by three PAL isoforms. These isoforms possessed similar native and subunit Mr, but were different in their pI, Km for Phe and optimal pH for activity. Also these were differentially induced and affected by metabolites belonging to particular branches of phenylpropanoid pathway.

Complex and shifting interactions of phytochromes regulate fruit development in tomato

Tomato fruit ripening is a complex metabolic process regulated by a genetical hierarchy. A subset of this process is also modulated by light signalling, as mutants encoding negative regulators of phytochrome signal transduction show higher accumulation of carotenoids. In tomato, phytochromes are encoded by a multi-gene family, namely PHYA, PHYB1, PHYB2, PHYE and PHYF; however, their contribution to fruit development and ripening has not been examined. Using single phytochrome mutants phyA, phyB1 and phyB2 and multiple mutants phyAB1, phyB1B2 and phyAB1B2, we compared the on-vine transitory phases of ripening until fruit abscission. The phyAB1B2 mutant showed accelerated transitions during ripening, with shortest time to fruit abscission. Comparison of transition intervals in mutants indicated a phase-specific influence of different phytochrome species either singly or in combination on the ripening process. Examination of off-vine ripened fruits indicated that ripening-specific carotenoid accumulation was not obligatorily dependent upon light and even dark-incubated fruits accumulated carotenoids. The accumulation of transcripts and carotenoids in off-vine and on-vine ripened mutant fruits indicated a complex and shifting phase-dependent modulation by phytochromes. Our results indicate that, in addition to regulating carotenoid levels in tomato fruits, phytochromes also regulate the time required for phase transitions during ripening.